lecture01

SYLLABUS: 

SYLLABUS BioBM 730/VetPR 731 Protein NMR Spectroscopy, Spring 2003 2 credits Linda K. Nicholson (LKN), Department of Molecular Biology and Genetics Robert E. Oswald (REO), Department of Molecular Medicine Wednesday, 2:00 - 3:40 PM, 271 Biotechnology Building

TEXTBOOK: 

TEXTBOOK title: Spin Dynamics: Basics of Nuclear Magnetic Resonance author: Malcolm H. Levitt publisher: John Wiley & Sons, Ltd. ISBN: 0-471-48922-0

TOPIC 1: Introduction: 

TOPIC 1: Introduction History of NMR NMR is Hard --- Why Bother? Advantages over other techniques Unique information

TOPIC 2: The Origin of the NMR Signal: 

TOPIC 2: The Origin of the NMR Signal What gives rise to an NMR signal in a protein? Spectroscopy Nuclear Spin Nuclear spin interactions Proteins and spin-1/2 nuclei Proteins and quadrupolar nuclei Two-dimensional NMR

TOPIC 3: One-dimensional protein NMR (1H, 13C, 15N): 

TOPIC 3: One-dimensional protein NMR (1H, 13C, 15N) The Classical description Introduction to Quantum Mechanics Product Operator description Standard Water Suppression Techniques TOPIC 4: Two-dimensional NMR

TOPIC 5: Hardware Principles: 

TOPIC 5: Hardware Principles The Superconducting Magnet The Probe The Console (transmitter, receiver, etc.)

TOPIC 6: Data Processing Principles: 

TOPIC 6: Data Processing Principles The Fourier Transform Maximizing the information content Zero Filling Apodization Phasing Baseline Correction

Topic 7: A “First Look” at a Protein - The 15N-1H HSQC spectrum: 

Topic 7: A “First Look” at a Protein - The 15N-1H HSQC spectrum What can we learn from the spectrum? Folded vs. Unfolded Correlation with hydrogen bond length Correlation with secondary structure Use in detecting perturbations (e.g. ligand binding) Fast exchange vs. slow exchange How does the experiment work? Detailed look at INEPT transfer Detailed look at Preservation of Equivalent Pathways The Use of Pulse Field Gradients Basic Principles of PFGs Use in Water Suppression Use in preservation of magnetization

Topic 8: 3D and 4D NMR: 

Topic 8: 3D and 4D NMR Principles Example Pulse Sequence Flow of magnetization Analysis using Product Operator Formalism

Topic 9: Solving the Resonance Assignment Problem: 

Topic 9: Solving the Resonance Assignment Problem Homonuclear Approach 2D-TOCSY 2D-NOESY 15N-only Approach 15N-1H-HSQC as “map” 3D-TOCSY to define spin systems 3D-NOESY to connect spin systems Triple Resonance Approach Backbone Assignments Side chain assignments Stereospecific assignments

Slide11: 

Topic 10: Structure Determination Topic 11: Relaxation and the Global and Internal Motions of Proteins Topic 12: Techniques for Larger Proteins

Lecture 1INTRODUCTION TO NMR: 

Lecture 1 INTRODUCTION TO NMR Linda K. Nicholson Jan. 22, 2003

This has 2 meanings – 1. NMR does not destroy the sample2. This course (and the book in progress) will be presented from the perspective of the protein, not from the perspective of pure NMR theory. : 

This has 2 meanings – 1. NMR does not destroy the sample 2. This course (and the book in progress) will be presented from the perspective of the protein, not from the perspective of pure NMR theory. NMR: A Kinder, Gentler Approach

History of Protein NMR : 

History of Protein NMR NMR is a relatively “new” technique compared with x-ray X-ray structure of myoglobin (17.8 kDa) by John Kendrew, 1963 Kendrew, J. C. (1963) Science 139, 1259-1266. Myoglobin and the structure of proteins. X-ray structure of hemoglobin (66 kDa) by Max Perutz, 1963 Perutz, M. F. (1963) Science 140, 863-869. X-ray analysis of hemoglobin. NMR structure of proteinase inhibitor IIA (6 kDa), 1985 Williamson, M. P., Havel, T. F. and Wuthrich, K. (1985) J. Mol. Biol. 182, 295-315. Why did NMR take so long to “evolve”?

Protein NMR Involves Several Different Disciplines: 

Protein NMR Involves Several Different Disciplines Hardware development Fourier transform, multiple-dimensional spectroscopy* Water suppression Recombinant DNA technology Experimental (pulse sequence) development* *Nobel Prize Awarded to Richard Ernst, 1991 + Nobel Prize Awarded to Kurt Wüthrich, 2002

Hardware Development: 

Hardware Development Radio Frequency (RF) Electronics Strong Magnets Computers

Hardware Development: 

Hardware Development Radio Frequency (RF) Electronics Magnets Computers

Who Invented Radio?: 

Who Invented Radio? Nikola Tesla? Tesla coil, 1891 Remote-controlled boat, 1898 US patents for fundamental radio technologies, 1900 The Great Radio Controversy http://www.pbs.org/tesla/ll/ll_whoradio.html

The Tesla Coil: 

The Tesla Coil

Tesla’s vision for wireless communication: 

Tesla’s vision for wireless communication Wall Street financier J. P. Morgan Promotional illustration for Tesla's "World System" Tesla's tower with dome frame. Completed in 1904.

Slide21: 

Guglielmo Marconi? Nobel Prize, 1909 Patent in England for crude wireless telegraphy device, 1896 US patent for “invention of radio”, 1904 The Great Radio Controversy

US Supreme Court Decision: 

US Supreme Court Decision Based on patent records and scientific publications, in June of 1943 the court decided that Nikola Tesla had invented modern radio technology. Tesla died January 7, 1943, alone and destitute in a New York hotel room.

Hardware Development: 

Hardware Development Radio Frequency (RF) Electronics Magnets Computers … some magnet details later!

Protein NMR Involves Several Different Disciplines: 

Protein NMR Involves Several Different Disciplines Hardware development Fourier transform, multiple-dimensional spectroscopy* Water suppression Recombinant DNA technology Experimental (pulse sequence) development* *Nobel Prize Awarded to Richard Ernst, 1991 + Nobel Prize Awarded to Kurt Wüthrich, 2002

Fourier transform, multi-dimensional spectroscopy : 

Fourier transform, multi-dimensional spectroscopy Other forms of spectroscopy measure spectrum using CW (continuous-wave) Monochromatic EM wave

Fourier transform, multi-dimensional spectroscopy : 

Fourier transform, multi-dimensional spectroscopy In the 1970s, pulse Fourier spectroscopy was developed.

Fourier transform, multi-dimensional spectroscopy : 

Fourier transform, multi-dimensional spectroscopy Allows simultaneous excitation of range of frequency space With multi-channel RF transmitters and receivers, allows multi-dimensional spectroscopy

The Great 2D NMR Controversy: 

The Great 2D NMR Controversy Jean Jeener, Lecture at Ampère Summer School, Basko Polje, Yugoslavia – 1971 Proposed to introduce a true second frequency dimension Proposed a two-pulse experiment in the time-domain for which Fourier transformation of the response would yield a 2D spectrum (unpublished) Richard Ernst, published the first 2D experiments W. P. Aue, E. Bartholdi and R. R. Ernst (1976) J. Chem. Phys. 64, 2229. Who Invented 2D NMR?

Protein NMR Involves Several Different Disciplines: 

Protein NMR Involves Several Different Disciplines Hardware development Fourier transform, multiple-dimensional spectroscopy* Water suppression Recombinant DNA technology Experimental (pulse sequence) development* *Nobel Prize Awarded to Richard Ernst, 1991 + Nobel Prize Awarded to Kurt Wüthrich, 2002

Why worry about water?: 

Why worry about water? Concentration of water in an aqueous solution is ~100 M Concentration of protein is typically 1 mM! Severe dynamic range problem (factor of 100,000)!!! Originally it was thought that NMR of proteins in aqueous solution was not possible …

Protein NMR Involves Several Different Disciplines: 

Protein NMR Involves Several Different Disciplines Hardware development Fourier transform, multiple-dimensional spectroscopy* Water suppression Recombinant DNA technology Experimental (pulse sequence) development* *Nobel Prize Awarded to Richard Ernst, 1991 + Nobel Prize Awarded to Kurt Wüthrich, 2002

2002 Nobel Prize in ChemistryKurt Wüthrich: 

2002 Nobel Prize in Chemistry Kurt Wüthrich Showed that NMR was possible for proteins

NMR is Hard --- Why Bother?: 

NMR is Hard --- Why Bother? Specific advantages No crystal packing artifacts In aqueous solution (natural environment) Conformational ensemble view Titrations (pH, ligands, etc.) are simple Unique information Global and internal dynamics Protonation states (pKas) can be determined Perturbations of chemical environment can be rapidly mapped

Protein NMR – What we need to know: 

Protein NMR – What we need to know For Structure: Amino acid sequence Resonance assignments (1H, 13C, 15N) Experimentally-derived structural constraints Structural model consistent with constraints For Dynamics: All of above Relaxation rates for individual nuclei of interest Model of motion consistent with relaxation rates

ASSIGNMENT FOR NEXT WEEKDownload Richard Ernst’s Nobel Lecture (as PDF file) from the following Web site:http://www.nobel.se/chemistry/laureates/1991/ernst-lecture.html : 

ASSIGNMENT FOR NEXT WEEK Download Richard Ernst’s Nobel Lecture (as PDF file) from the following Web site: http://www.nobel.se/chemistry/laureates/1991/ernst-lecture.html 1. Read the lecture. 2. Make a list of terms and/or concepts that you do not yet understand (but would like to!). 3. Bring the list to class next Wednesday to share with the class.